Asthma has increased in prevalence and severity in the industrialized world for the past two decades; it is the most common chronic illness of children and has had a rising mortality rate. The "hygiene hypothesis" proposes that reduced early-life exposures to microbes and microbial products are responsible for this epidemic. According to this paradigm, microbial exposures inhibit the development of Th2-skewed responses to environmental allergens, putatively through the induction of Th1 and/or regulatory-type responses. The epidemiological data supporting this hypothesis are compelling, if correlative: children raised in a farm environment (especially with significant exposure to livestock), with increased numbers of older siblings or early daycare attendance (and thus exposed to childhood infections at an earlier age), or with early life exposure to pets have a reduced risk of developing asthma and atopic disorders. The hygiene hypothesis has been difficult to directly test in humans; it remains important to identify the pathways that promote immune tolerance among microbe-exposed children. Understanding the mechanisms of this tolerance will aid development of novel tolerance-inducing therapeutic approaches, and thus reduce the burden, of asthma and atopy. One candidate for inducing tolerance is endotoxin; endotoxin levels in environmental dust correlate with reduced risk of atopy in some studies. Nevertheless, endotoxin inhalation has not been shown to protect against atopic disease in humans; indeed, much evidence points to a deleterious effect of endotoxin in asthma. A second candidate is bacterial (CpG-rich) DNA. We have previously demonstrated, using murine models, that oligonucleotides containing CpG-centered sequence motifs (CpG ODN) are highly effective in both preventing and reversing atopic airway disease. These compounds can suppress allergen-specific immune (Th2-type) responses and induce Th1-type as well as regulatory (IL-10) cytokine responses. CpG-ODN mimics the immunostimulatory effects of native bacterial DNA. Like endotoxin, bacterial DNA is recognized by a toll-like receptor (TLR-9) and potently activates the innate immune system. We suggest that exposure to bacterial DNA may mediate protection against atopy. Thus, we propose to test the hygiene hypothesis, and to compare the modulatory effects of endotoxin and CpG-rich DNA. The overarching hypothesis driving these studies is that early-life exposure to microbial products promotes tolerance and reduces later susceptibility to developing atopic disorders such as asthma. Inhalation of endotoxin and bacterial DNA may influence response to inhaled allergens through different mediators and pathways; these effects may be additive, synergistic, or even antagonistic. We propose to address these hypotheses in the following Aims: Aim 1: To test the hypothesis that early-life exposure to microbial products (bacterial DNA or endotoxin) reduces later susceptibility to developing atopic disorders. Question 1A: Does early life exposure to microbial products alter the pulmonary inflammatory milieu? Question 1B: Does early life exposure to microbial products alter later susceptibility to developing atopic asthma? Question 1C: Is early-life exposure to antigen as well as microbial products required for antigen-specific tolerance? Aim 2: To test the hypothesis that early-life exposure to microbial products promotes immune tolerance by inducing a regulatory cell population. Question 2A: What mediators promote immune tolerance induced by early life exposure to microbial products? Question 2B: What cells are responsible for immune tolerance induced by early life exposure to microbial products? [unreadable] [unreadable]